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Catalytic activity of ethylene-bridged aminoacid-copper(II) complexes for the dismutation of superoxide
J’olyhe&on Vol. 4, No. 12, pp. 2031-2038, Printed in Great Britain
1985 0
0277-5387/85 S3.00+ .OO 1985 Pagamon Press Ltd
CATALYTIC ACTIVITY OF ETHYLENE-BRIDGED AMINOAClD-COPPER(II) COMPLEXES FOR THE DISMUTATION OF SUPEROXIDE B. M. KATZ and V. I. STENBERG* Department of Chemistry, University of North Dakota, Grand Forks, ND 58202, U.S.A. (Received 2 February 1985; accepted after revision 10 June 1985)
Abstract-Copper complexes of bridged ethylene derivatives of the aminoacids leucine, isoleucine, valine, alanine, glycine and 2-methylalanine showed marked differences in their ability to quench superoxide. Leucine, alanine and 2-methylalanine ethylene-bridged copper complexes displayed the highest activity, the glycine complex was the least active, and valine had low and isoleucine intermediate activities. A study of differences in speciation, chelation, electronic and steric factors with the aid of molecular models led to a tentative hypothesis that the methyl groups of isoleucine and valine, when distorted toward tetrahedral, are able to block the axial site of copper from binding to superoxide. The glycine-bridged copper complex is believed to form a dimer at the axial copper site which completely blocks the attachment of superoxide.
Copper is an essential trace element required by several enzymes of the body. One of these is superoxide dismutase which has the function of inhibiting the superoxide anion (0;). The production of the latter has been associated with oxygen toxicity.’ The active site of the enzyme is copper and the mechanism of catalytic activity of copper towards superoxide appears to involve opencoordination positions of copper for reaction with superoxide.* Kinetic studies of superoxide dismutation by copper histidine,3 o-phenanthroline4 and an ESR study of copper salicylate’ indicate that superoxide initially binds to the axial copper site. When the open-coordination site of copper is bound by a polypeptide nitrogen, the activity of the metal complex is markedly reduced.6 Aminoacid-copper complexes were found to exhibit a wide range of activities with superoxide, but mechanistic studies to elucidate structure-activity relationships were not done.’ The objectives of this study were to synthesize various substituted ethylenediaminediacetic acid complexes and examine their ability to react with the superoxide ion. The ethylene bridge is for the purpose of attaining compounds that possess enhanced chelation properties compared to the * Author to whom correspondence
should be addressed.
unbridged amino acids.* In catabolism, the organic ligand is expected to yield the corresponding ketoacids and ethylenediamine by transamination. The ethylenediamine ligands are derivatives of the aliphatic aminoacids : valine, isoleucine, leucine, alanine, 2-methylalanine and glycine. EXPERIMENTAL Aminoacids and ethylenediamine-iV,N’-diacetic acid were supplied by Sigma Chemical Co., Eastman Co. and Fisher Scientific Co. NMR spectra were recorded on a Varian EM 390 spectrometer. The’ aminoacid derivatives were dissolved in NaOD. The internal standard was 3-(trimethylsilyl)-lpropanesulfonic acid, sodium salt hydrate. General procedure for the preparation of ethylene derivatives of aminoacids
The appropriate aminoacid (0.1 mol) was dissolved in 100 cm3 of water containing 0.1 mol NaOH and 0.05 mol sodium carbonate. To this solution was added 0.05 mol ethylene bromide in 100 cm3 95% ethanol. The solution was heated for approximately 3 h under reflux or until a homogeneous solution was attained. This solution was cooled and acidified to pH 3 with 6 M HCl. The resultant precipitate was filtered, washed with
2031
2032
B. M. KATZ and V. I. STENBERG
distilled, deionized water and 95% ethanol. The aminoacid derivatives were air-dried. Solutions that did not precipitate with acid were concentrated until a precipitate formed. The compounds were purified by dissolving in aqueous solution, pH 10, and reprecipitating by adding HCl to pH 3. n,r-leucine-ethylene. Yield 18.3x, m.p. (“C) 237-239 decomposed (d), molecular weight (MW) 288.22. Found: C, 58.2; H, 9.4; N, 9.4. Calc. for Cr,H,,O,N,: C, 58.3; H, 9.7; N, 9.7%. NMR spectra : 6 0.9 (d, 6, J = 2 Hz) ((X,)&H ; 1.4 (m, 3) CH,CH; 2.6 (s, 2) CHzCHz; 3.1 (t, 1, J = 4 Hz) CHCOO-. D,t-isoleucine-ethylene. Yield 21x, m.p. (“C) 260262 d, MW 288.22. Found: C, 58.3; H, 10.0; N, 9.8. Calc. for C,,H,,0,N2: C, 58.3; H, 9.7; N, 9.7. NMR: 6 0.08 (t, 6) CH,CH,, CH$H; 1.1 (m, 2) CH,CH ; 1.5 (m, 1) CH&H; 2.5 (s, 2) CH&H, ; 2.8 (d, d, 1, J = 3 Hz, 6 Hz) CHCOO-. L-v&w-ethylene. M.p. (“C) 301-302 d, reported 270-310 d.’ NMR: 6 0.9 (d, d, 6, J = 1 Hz), (CH&CH; 1.8 (m, 1, J = 4 Hz) CHCHz; 2.7 (s, 2) CH,CH2 ; 2.9 (d, 1, J = 6 Hz) CHCGO-. w-&mine-ethylene. M.p. (“C) 260-262 d, rep-. orted 264266.” NMR: 6 1.4 (d, 3, J = 4.5 Hz), CH$H; 2.9 (s, 2) CHzCHz ; 3.3 (q, 1, J = 5 Hz) CHCOO-. The method of Schlesinger” was used for the preparation of 2-methylalanine. Test solutions
Solutions for superoxide-quenching assays, ESR and photometric studies were prepared by weighing an appropriate amount of the ligand to obtain 1 x 10V2 M aminoacid-ethylene derivatives. A low2 M solution of CU(NO~)~ was also prepared. Dilutions of 1 x low3 and 1 x lop4 M copper complexes were attained by mixing equal volumes of the 1 x lo- 2 M solution of the ligand and 1 x 10m2 M solution of the copper ion and diluting with a pH-7.6 aqueous buffer. Only a metal : ligand ratio of 1: 1 was prepared. Superoxide assays
K02 method.” To a series of test tubes were added 3.0 cm3 buffer solution followed by 0.05,0.1,0.3 and 0.5 cm3 of 1 x 10e4 M solution of the compound to be tested and brought to the same volume with 0.45, 0.4, 0.2 and 0 cm3 of distilled deionized water. The final concentration varies from 1 x low6 to 1 x lo-’ M. To each tube was then added 0.1 cm3 of a nitroblue tetrazolium (NBT) solution (4 mg cmw3) in dimethylsulfoxide (DMSO) (Aldrich Gold Label), 0.1 cm3 of a solution containing 20 mg KO, with 0.1056 g/10 cm3 18-crown-6 (Aldrich) in DMSO. The tubes were shaken thoroughly after the addition
of each reagent. The absorbance was read immediately at 540 rnp using a Bausch & Lomb Spectronic 20. All readings were taken against a blank containing everything except the potassium superoxide solution. A standard curve of increasing concentrations of K02 with absorbance gave a straight line with a correlation coefficient of 0.999. Three repetitive assays for one concentration were conducted for most of the test compounds in order to assure reproducibility. The average error did not exceed 5%. /?-Nicotinamide-adeninedinucleotide (NAW method.13 The assay was identical to the K02
method except for the following. After the addition of the NBT solution, 0.1 cm3 phenazine methosulfate (PMS, 10 mg/lOO cm3 in buffer) was added. The reaction was then started by the addition of 0.3 cm3 of NADH (1.5 mM in buffer) solution. The NADH solutions were made fresh before each assay. After standing in the dark for 10 min the readings were taken as described above. Preweighed vials purchased from Sigma Chemical Co. were the source of NADH. The results of three successive determinations with NADH from the same vial did not vary over 1%. The phosphate buffer was prepared by mixing 4.24 cm3 of solution A (22.6 g NaH,PO, * Hz0 in 11) and 45.75 cm3 of solution B (71.7 g Na,HPO,* 12H20 in 1 1) and diluting to 200 cm3. Distilled, deionized water was used for all preparations and assays. Potentiometric ments
and spectrophotometric
measure-
Potentiometric titrations were performed on the valine-, leucin* and isoleucinmpper complexes with metal : ligand ratio of 1: 1, 10e2 M concentration, 25”CandO.l M KN03.‘4Thedissociationconstants were calculated from the inflection points. With the copper complexes of valine, leucine and isoleucine ethylene-bridged compounds, dissolution does not occur until a slightly basic pH is reached. Rapid back-titration with HCI provided a means for titration to an acid pH before bulk precipitation occurred. The values obtained with the backtitration were used in the calculations. Titration of copper with the basic ligands for determination of stability constants were done as outlined for nickel glycine. l4 The calculation of association constants was adapted to a dibasic acid system as described by Courtney et al.’ ’ Equations (l)-(5) were assumed for the calculation of the dissociation constants where [L2 -1 is the concentration of completely deprotonated ligand, [LH-] is the concentration of monopronated ligand, [LH,] is the concentration of protonated
2033
Catalytic activity of ethylene-bridged aminoacid-copper(II) complexes NADH
ligand, and at- is the fraction of the deprotonated ligand : LH2 e LH- +H+,
(1)
LH- P L2- +H+,
(2)
k, = CLH-lCH+l D-b1 ’
(3)
klkz
z- -
aL
-
(5)
[H+]2+ki[H+]+k1k2’
The calculation of the metal-ligand speciation was done using eqns (6)-(S), where CL’-] = L, x a;-, CL=]is the original concentration and K1 and K2 are the association constants of metal and ligand : 1
B‘“‘+ = 1+K2[L2-]+KlK2[L2-]2
‘“t---2
&CL’-1 1+K,[L2-]+K,K2[L2-]2’
K,K2[L2-]2 =
1 +Kl[L2-]
M
’
fraction of CuHL+ ; (7) BCUL
IO IO-’
Fig. 1. The quenching of superoxide by the copper complexes using the NADH assay. (- ) Ethylenedileucine, (---) ethylene-di-isoleucine, and (--a-) ethylene-divaline.
fraction of free Cu2 + ; (6)
BcuLH+ =
4 6 6 Concentration
+K,K,[L’-1” fraction of CuL.
(8)
Calculations for speciation were made for a ligand concentration of 1 x 10m5 M similar to that for the superoxide-quenching assays. Calculations made for the alanine- and glycine-bridged copper complexes from the constants given by Chaberek and Martell16 indicate that both compounds contained less than 0.1% of the free Cu(I1). Calculations of metal-ligand association constants followed the procedure of Courtney et al. I5 for dibasic acids. All absorbance measurements were made with a Beckman 25 recording spectrophotometer at a pH of 7.6.
RIBULTS
The rates of reaction of the complexes with superoxide ion in vitro were leucine r alanine r 2methylalanine > isoleucine > valine >>glycine (cf. Figs l-4). The ligands without copper had no reactivity with the superoxide anion. Copper(I1) ion in the absence of the synthesized aminoacid ligands quenched superoxide at rates comparable to that of the leucine complex. The activity to quench the superoxide ion was examined at four different
K02
90
00 70 60 50
ESR spectroscopy
Copper complexes in aqueous solution buffered at pH 7 and ambient temperature were taken in capillaries with the concentration of the metal and ligand each at 10m3 M. Superoxide was prepared immediately before use as described above. Ratios of l:l, 1:2, 1:4, 1:6 and 1:8 (metal complex : superoxide) were prepared. Measurements were taken on a Model ER-420 JOEL X band ESR spectrometer.
40 30 20
2
4 6 6 Concentration
IO 10m6 M
Fig. 2. The quenching of superoxide by the copper Ethylene complexes using the KO, assay. (-) dileucine, (---) ethylene-d&isoleucine, (-.-) ethylenedivaline, and (. . .) Cuz+.
2034
B. M. KATZ and V. I. STENBERG NADH
I
I
I
2
I
I
I
4
6
8
Concentrotlon
2
3
4
5
IO 10-e
6
7
8
9
1011
PH
I
M
Fig. 3. The quenching of superoxide by the copper ) Ethylenecomplexes using the NADH assay. (ethylene-diglycine, and (-.-) dialanine, (---) ethylene-2-methylalanine.
Fig. 5. Species distribution for the 1: 1 copper(II)ethylerwdileucine complex as a function of pH. (a) Cuz+, (b) CuHL+, and (c) CuL.
IO_
0
9-
of the copper complexes. Although the reactivity order of the copper complexes were the same for the two independent assays, the potassium superoxide assay sensitivity decreased markedly at higher concentrations of the complexes. The results in Figs 5-7 indicate the speciation is the same for the three different copper complexes. The main species in the region of pH 7-8 for the valine-, leucine- and isoleucine-ethylene-copper complexes ia a copper complex with a net plus one charge. The titration curves of the ethylene-bridged amino acids (unprocessed data for Figs 5-7) shows .. two inflection points. concentrations
G 8E 2 u (;
765-
& 4ae 32II2
3
4
5
6
7
8
9
1011
PH
Fig. 6. Species distribution for the 1: 1 copper(IIb ethylendi-isoleucine complex as a function of pH. (a) Cu’+, (b) CuHL+, and (c) CUL.
2
4
6
Concentration
8
IO 10e6
M
Fig. 4. The quenching of superoxide by the copper ) Ethylenecomplexes using the KOs assay. (dialanine, and (--) ethylene-diglycine.
Fig. 7. Species distribution for the 1: 1 copper(II)ethylentiivaline complex as a function of pH. (a) Cu’+, (b) CuHL+, and (c) CuL.
Catalytic activity of ethylene-bridged aminoacid-copper(II) complexes
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Table 1. The UV and visible spectra of substituted ethylenediaminediacetic acid-copper complexes in aqueous solution Visible Copper-ethylene complex
1lppX (nm)
Valine Isoleucine Leucine Alanine Glycine 2-Methylalanine
635665 630-665 640-665 66&700 60&615 580620
uv
E,, 39 31 27 30 16 19
$4 254 250 250 250 nd” -*
E,, 4465 4135 3750 4000
‘Not detected. *Not determined. M
The visible and UV spectra from aqueous IOOG solutions of the bridged aminoacid-copper comFig. 8. ESR spectra of ambient aqueous solutions of plexes were determined in order to observe possible copper(I1) complexes. (a) Ethylene-N,N’-dialanine, (b) differences in configuration. The A,,,, and E,, are ethylenediglycine; (c) ethylen&V,N’-di-isoleucine, and summarized in Table 1. The visible region of the (d) ethylene-dileucine. spectra consists of broad absorption bands for all complexes. They are very close in energy but differ in intensity. The latter increase in the series : leucineDISCUSSION < isoleucine- < valine-copper complexes. The rZ, The substituted ethylenediaminediacetic acidfor the copper-alanine complex was slightly higher than the other copper complexes in the series. In the copper complexes formed from leucine, alanine and UV region of the spectra, very intense peaks at low 2-methylalanine display the highest rate of reactivity concentrations were observed at the 250~nm region with the superoxide ion of the derivatives for all complexes but the glycine-copper complex. synthesized (Figs l-4). The copper-glycine derivatThis peak was absent with copper and the ligands ive did not show any activity. In these complexes, the presence of the copper ion is essential to the without copper. complexes in order for them to react with the The ESR spectra of the copper-aminoacid bridged complexes were obtained in the presence superoxide anion. A superoxide intermediate is and absence of superoxide. All complexes except the believed to account for observations in kinetic glycine had four hyperfine peaks. The glycine one studies of superoxide reacting with copper comhad seven peaks. Figure 8 shows the spectra of plexes. 4~17 The site proposed for superoxide alanine-, glycine-, isoleucine- and leucint+copper attachment is the axial site of Cu(II), and the complexes before superoxide was added. The mechanism of the interaction is represented by eqns spectrum of the copper-alanine complex was poorly (9)-(11): resolved compared to the other copper complexes. Cu(I1) + 0; = Cu(II)O,, (9) After the first addition of superoxide anion, another type of spectra occurs which represents a cu(II)o; = Cu(I)O,, (10) paramagnetic species but differs from the spectrum Cu(I)O z*Cu(I)+O,. (11) without superoxide by an increase in hyperfine structure. This occurs before further reduction to a Cu(II)02 and Cu(I)02 are the oxygen adducts. species where all absorption bands disappear with The ESR spectra of solutions in which these further addition of superoxide. complexes are reacted with various concentrations The spectra for the copper-valine complex before of the superoxide anion give evidence for the initial and after addition of two increments of superoxide is bonding of the anion with the copper(I1) cation and illustrated in Fig. 9. The copper-valine complex its subsequent reduction to copper(I). The buffered, prior to superoxide addition did not have a aqueous, ambient-temperature ESR spectra of all pronounced hyperfine structure compared to the copper complexes show an initial increase in the leucine one (Figs 8 and 9). hyperflne structure of copper with increasing
2036
B. M. KATZ and V. I. STENBERG
c
I IOOG
Fig. 9. ESR spectra of ambient aqueous solutions of ethyleneN,N’-divaline with increments of superoxide. (a) No equivalent superoxide, (b) one equivalent superoxide, and (c) three equivalents of superoxide.
increments of superoxide followed by loss of the hyperflne structure as reduction to Cu(1) occurs. As an example, the ESR spectra of the valine derivative copper complex interaction with three different concentrations of superoxide are given in Fig. 9. In a study of copper salicylate, DeAlvare et d5 previously interpreted such spectra as superoxide complexing to copper with subsequent reduction. All five complexes examined herein have two nitrogen atoms complexed with the copper ion. Agarwal and Perrin I* have shown that the number of nitrogen atoms bound to copper can be distinguished by the visible spectra. If an absorption occurs below 600 nm, the copper ion is bound to three nitrogen atoms, but above 600 nm to two nitrogen atoms. The compounds in the present study all have absorption maxima above 600 nm (Table 1). The intense peaks in the UV spectra of the copper complexes are charge-transfer bands of the N-Cu-N bonds as verified by their absence in the spectra of copper solutions and the ligands without copper. By comparison with the bis(diamine)copper complexes, the bands are assigned to a c-o* (R,N-Cu-NR,) transition.” Only the glycine complex does not show the chargetransfer band. This indicates it has a different structure from the other four aminoacid complexes. Since the concentration of copper is identical for all the complexes, the data of Figs l-4 demonstrate that the copper ligand nature influences the rate of reaction with the superoxide anion. The coordination number of the copper ion does not change within the series: isoleucine, leucine, and valine ethylene-bridged copper complexes. The speciation
curves illustrated in Figs 5-7, calculated from the experimentally derived association constants for these three complexes, are virtually identical. Consequently, the diverse reactivities of these three complexes towards the superoxide ion are caused by factors other than speciation. Steric hindrance to the approach of the superoxide ion to the copper(I1) is believed to account for the decreased reactivity of the valine- and isoleucinebridged derivatives. Both have branching of the hydrocarbon chain at C-3, whereas the most reactive three complexes do not. Hence there is steric hindrance in close proximity to the active site. The 2methylalanine-bridged complex ability to quench superoxide approximates that of the leucinecomplex in spite of the extra methyl group at C-2. Therefore, the important stericinteractions appear to be at C-3. By using conformational analysis, Raos and Simeon2’ presented evidence that a methyl group which extends over copper reduces hydration of the axial copper site. This, together with the known tendency of copper complexes to distort toward tetrahedral, provides the possibility of the two C-3 alkyl groups of isoleucine and valine to extend over the axial site ofcopper to block this site and lessen its catalytic activity. 21-26 This was examined with the aid of molecular models. The C-3 alkyl groups of the isoleucine and valine complexes are unlikely to extend over copper in a square planar conformation. However, with an incease in distortion toward tetrahedral, these alkyl groups can readily be positioned over the copper(H). There is evidence for tetrahedral distortion among the six ethylenediamineacetic acid complexes of
Catalytic activity of ethylene-bridged aminoacid-copper(II) complexes copper studied herein. Red shifts and intensity enhancements of d-d electronic absorption bands accompany changes from square planar toward tetrahedral geometry.‘r The I, of the aminoacid bridged complexes (Table 1) have broad bands and are close in energy, but the intensities increase markedly from leucine- to valine-bridged copper complexes. With the exception of alanine, the degree of tetrahedral distortion roughly corresponds to the extent of superoxide quenching by these complexes. It appears that both the presence of two alkyl blocking groups at C-3 and tetrahedral distortion are necessary and act synergistically to block the axial site of copper(I1) to superoxide. Bereman et ~1.~’ concluded that tetrahedral distortion is the single most important dominating factor which changes Er12for their copper complexes and ESR parameters which reflect the degree of tetrahedral distortion varied linearly with E1,2.27 Yokoi and Addison” also showed that ESR parameters varied linearly with E,,,. It is therefore probable that the reduction potential varies in the series studied here. However, it appears that the resultant potential is a function of the structure which determines the availability of the axial copper site for the redox reaction with superoxide. The lack of activity of the copper glycine-bridged complex is attributed to formation of a dimer on the axial copper site. The complex is the smallest of the series, and small copper compounds have been found to dimerize.2g In an analogous study of copperaminoacid complexes, only with glycine did a third group add to the axial site of copper.30 Although dimeric copper compounds are antiferromagnetitally coupled, resulting in an absence of an ESR spectra,31 the ESR of the copper-glycine-ethylene
H H
1
‘c-c
H 1,H
2037
complex had seven peaks, indicating a weaker interaction of two copper nuclei (cf. Fig. 8). Although it was recognized that dimerization had occurred from a frozen ESR spectra of a 1: 1 copper-bridged glycine complex, the nature of the structure was not forwarded.32 The significance of seven lines for a copper dimer was attributed to out-ofplane interactions for the N,N’-ethylenebis(salicyldeneiminato) copper complex.33 Based on the ESR spectrum and the absence of a chargetransfer band which indicates a change of symmetry (Table l), a tentative structure is proposed (Fig. 10). The axial copper site in this structure is occupied by another bridged glycine so that it is inaccessible to super-oxide, resulting in deactivation of the catalytic activity of copper to quench superoxide.
REFERENCES 1. I. Fridovich, Science 1978,201,875. 2. G. Rotilio, A. Rigo and L. Calabrese, Frontiers in Physicochemical Biology, p. 357. AcademicPress, New York (1978). 3. J. Weinstein and B. M. Bielski,J. Am. Chem. Sot. 1980, 120,4916.
4. S. Goldstein and G. Czapski, J. Am. Chem. Sot. 1983, 105,7276.
5. L. R. DeAIvare, K. Goda and T. Kimura, Biochem. Biophys. Res. Commun. 1976,69,687.
6. C. Amar, E. Vilkas and J. Foos, J. Znorg. Biochem. 1982,17,313.
7. K. E. Joester, G. Jung, U. Weber and U. Weser, FEBS L&t. 1972,25,25.
8. F. Bell and K. A. K. Lott, Modern Approaches to Inorganic Chemistry, p. 150. Butterworths, London (1963). 9. R. J. Magee, W. Mazurek, M. J. CSConner and A. T. Phillip, Aust. J. Chem. 1974,27, 1885. 10. P. Blondeau, C. Berse and D. Gravel, Can. J. Chem. 1967,45,49. 11. N. Schlesinger, Ber. Chem. Dtsch. Ges. 1911,44,1135. 12. M. Younes and U. Weser, FEBS Lett. 1976,61,209. 13. R. Fried, Biochemie 1975,57,657. 14. R. J. Angelici, Synthesis and Technique in Inorganic Chemistry, p. 115. W. B. Saunders, Philadelphia (1977). 15. R. C. Courtney, S. Chaberek, Jr and A. E. Martell, J. Am. Chem. Sot. 1953,75,4814. 16. S. Chaberek, Jr and A. E. Martell, J. Am. Chem. Sot. 1952,74,5052. 17. J. Rabini, D. Klug-Roth and J. Lillie, J. Phys. Chem. 1973,77,1169.
l-i
-H
Fig. 10. Proposed structure for Cu(II)-e.thylenf+diglycine.
18. R. P. Agarwal and D. D. Perrin, J. Chem. Sot., Dalton Trans. 1976,89. 19. B. P. Kennedy and A. B. P. Lever, J. Am. Chem. Sot. 1973,%, 6907. 20. N. Raos and V. I. Simeon, J. Znorg. Biochem. 1983,1l#, 133.
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B. M. KATZ and V. I. STENBERG
21. K. F. Purcell and J. C. Kotz, Inorganic Chemistry, p. 761. W. B. Saunders, Philadelphia (1977). 22. R. C. Rosenberg, C. A. Root, R. K. Bernstein and H. B. Gray, J. Am. Chem. Sot. 1975,97,2092. 23. L. Sacconiand M. Ciampolini, J. Chem. Sot. 1964,276. 24. M. J. Adam and L. D. Hall, Can. J. Chem. 1982,60, 2229. 25. H. Yokoi, Bull. Chem. Sot. Jpn 1974,47,3037. 26. S. Yamada and A. Tokeuchi, Coord. Chem. Rev. 1982,43, 187. 27. R. D. Bereman, J. R. Dorfman, J. Bordner, P. D. Rillema, P. McCarthy and G. D. Shields, J. Znorg. Biochem. 1982,16,47.
28. H. Yokoi and A. W. Addison, Znorg. Gem. 1977,16, 1341. 29. R. bterberg, Coord. Chem. Rev. 1974,12,309. 30. I. Nagypti, E. Farkas and A. Gergely, J. Znorg. Nucl. Chem. 1975,37,2145. 31. U. Weser, K. H. Sellinger, E. Lengfelder, W. Werner and J. Striihle, Biochim. Biophys. Acta 1980, 631, 232. 32. N. Yokoi and M. Chikera, J. Chem. Sot., Chem. Commun. 1982,1125.
33. G. 0. Carlisle and W. E. Hatfield, Znorg. Nucl. Chem. Z.&t. 1970,6, 633.
1985 0
0277-5387/85 S3.00+ .OO 1985 Pagamon Press Ltd
CATALYTIC ACTIVITY OF ETHYLENE-BRIDGED AMINOAClD-COPPER(II) COMPLEXES FOR THE DISMUTATION OF SUPEROXIDE B. M. KATZ and V. I. STENBERG* Department of Chemistry, University of North Dakota, Grand Forks, ND 58202, U.S.A. (Received 2 February 1985; accepted after revision 10 June 1985)
Abstract-Copper complexes of bridged ethylene derivatives of the aminoacids leucine, isoleucine, valine, alanine, glycine and 2-methylalanine showed marked differences in their ability to quench superoxide. Leucine, alanine and 2-methylalanine ethylene-bridged copper complexes displayed the highest activity, the glycine complex was the least active, and valine had low and isoleucine intermediate activities. A study of differences in speciation, chelation, electronic and steric factors with the aid of molecular models led to a tentative hypothesis that the methyl groups of isoleucine and valine, when distorted toward tetrahedral, are able to block the axial site of copper from binding to superoxide. The glycine-bridged copper complex is believed to form a dimer at the axial copper site which completely blocks the attachment of superoxide.
Copper is an essential trace element required by several enzymes of the body. One of these is superoxide dismutase which has the function of inhibiting the superoxide anion (0;). The production of the latter has been associated with oxygen toxicity.’ The active site of the enzyme is copper and the mechanism of catalytic activity of copper towards superoxide appears to involve opencoordination positions of copper for reaction with superoxide.* Kinetic studies of superoxide dismutation by copper histidine,3 o-phenanthroline4 and an ESR study of copper salicylate’ indicate that superoxide initially binds to the axial copper site. When the open-coordination site of copper is bound by a polypeptide nitrogen, the activity of the metal complex is markedly reduced.6 Aminoacid-copper complexes were found to exhibit a wide range of activities with superoxide, but mechanistic studies to elucidate structure-activity relationships were not done.’ The objectives of this study were to synthesize various substituted ethylenediaminediacetic acid complexes and examine their ability to react with the superoxide ion. The ethylene bridge is for the purpose of attaining compounds that possess enhanced chelation properties compared to the * Author to whom correspondence
should be addressed.
unbridged amino acids.* In catabolism, the organic ligand is expected to yield the corresponding ketoacids and ethylenediamine by transamination. The ethylenediamine ligands are derivatives of the aliphatic aminoacids : valine, isoleucine, leucine, alanine, 2-methylalanine and glycine. EXPERIMENTAL Aminoacids and ethylenediamine-iV,N’-diacetic acid were supplied by Sigma Chemical Co., Eastman Co. and Fisher Scientific Co. NMR spectra were recorded on a Varian EM 390 spectrometer. The’ aminoacid derivatives were dissolved in NaOD. The internal standard was 3-(trimethylsilyl)-lpropanesulfonic acid, sodium salt hydrate. General procedure for the preparation of ethylene derivatives of aminoacids
The appropriate aminoacid (0.1 mol) was dissolved in 100 cm3 of water containing 0.1 mol NaOH and 0.05 mol sodium carbonate. To this solution was added 0.05 mol ethylene bromide in 100 cm3 95% ethanol. The solution was heated for approximately 3 h under reflux or until a homogeneous solution was attained. This solution was cooled and acidified to pH 3 with 6 M HCl. The resultant precipitate was filtered, washed with
2031
2032
B. M. KATZ and V. I. STENBERG
distilled, deionized water and 95% ethanol. The aminoacid derivatives were air-dried. Solutions that did not precipitate with acid were concentrated until a precipitate formed. The compounds were purified by dissolving in aqueous solution, pH 10, and reprecipitating by adding HCl to pH 3. n,r-leucine-ethylene. Yield 18.3x, m.p. (“C) 237-239 decomposed (d), molecular weight (MW) 288.22. Found: C, 58.2; H, 9.4; N, 9.4. Calc. for Cr,H,,O,N,: C, 58.3; H, 9.7; N, 9.7%. NMR spectra : 6 0.9 (d, 6, J = 2 Hz) ((X,)&H ; 1.4 (m, 3) CH,CH; 2.6 (s, 2) CHzCHz; 3.1 (t, 1, J = 4 Hz) CHCOO-. D,t-isoleucine-ethylene. Yield 21x, m.p. (“C) 260262 d, MW 288.22. Found: C, 58.3; H, 10.0; N, 9.8. Calc. for C,,H,,0,N2: C, 58.3; H, 9.7; N, 9.7. NMR: 6 0.08 (t, 6) CH,CH,, CH$H; 1.1 (m, 2) CH,CH ; 1.5 (m, 1) CH&H; 2.5 (s, 2) CH&H, ; 2.8 (d, d, 1, J = 3 Hz, 6 Hz) CHCOO-. L-v&w-ethylene. M.p. (“C) 301-302 d, reported 270-310 d.’ NMR: 6 0.9 (d, d, 6, J = 1 Hz), (CH&CH; 1.8 (m, 1, J = 4 Hz) CHCHz; 2.7 (s, 2) CH,CH2 ; 2.9 (d, 1, J = 6 Hz) CHCGO-. w-&mine-ethylene. M.p. (“C) 260-262 d, rep-. orted 264266.” NMR: 6 1.4 (d, 3, J = 4.5 Hz), CH$H; 2.9 (s, 2) CHzCHz ; 3.3 (q, 1, J = 5 Hz) CHCOO-. The method of Schlesinger” was used for the preparation of 2-methylalanine. Test solutions
Solutions for superoxide-quenching assays, ESR and photometric studies were prepared by weighing an appropriate amount of the ligand to obtain 1 x 10V2 M aminoacid-ethylene derivatives. A low2 M solution of CU(NO~)~ was also prepared. Dilutions of 1 x low3 and 1 x lop4 M copper complexes were attained by mixing equal volumes of the 1 x lo- 2 M solution of the ligand and 1 x 10m2 M solution of the copper ion and diluting with a pH-7.6 aqueous buffer. Only a metal : ligand ratio of 1: 1 was prepared. Superoxide assays
K02 method.” To a series of test tubes were added 3.0 cm3 buffer solution followed by 0.05,0.1,0.3 and 0.5 cm3 of 1 x 10e4 M solution of the compound to be tested and brought to the same volume with 0.45, 0.4, 0.2 and 0 cm3 of distilled deionized water. The final concentration varies from 1 x low6 to 1 x lo-’ M. To each tube was then added 0.1 cm3 of a nitroblue tetrazolium (NBT) solution (4 mg cmw3) in dimethylsulfoxide (DMSO) (Aldrich Gold Label), 0.1 cm3 of a solution containing 20 mg KO, with 0.1056 g/10 cm3 18-crown-6 (Aldrich) in DMSO. The tubes were shaken thoroughly after the addition
of each reagent. The absorbance was read immediately at 540 rnp using a Bausch & Lomb Spectronic 20. All readings were taken against a blank containing everything except the potassium superoxide solution. A standard curve of increasing concentrations of K02 with absorbance gave a straight line with a correlation coefficient of 0.999. Three repetitive assays for one concentration were conducted for most of the test compounds in order to assure reproducibility. The average error did not exceed 5%. /?-Nicotinamide-adeninedinucleotide (NAW method.13 The assay was identical to the K02
method except for the following. After the addition of the NBT solution, 0.1 cm3 phenazine methosulfate (PMS, 10 mg/lOO cm3 in buffer) was added. The reaction was then started by the addition of 0.3 cm3 of NADH (1.5 mM in buffer) solution. The NADH solutions were made fresh before each assay. After standing in the dark for 10 min the readings were taken as described above. Preweighed vials purchased from Sigma Chemical Co. were the source of NADH. The results of three successive determinations with NADH from the same vial did not vary over 1%. The phosphate buffer was prepared by mixing 4.24 cm3 of solution A (22.6 g NaH,PO, * Hz0 in 11) and 45.75 cm3 of solution B (71.7 g Na,HPO,* 12H20 in 1 1) and diluting to 200 cm3. Distilled, deionized water was used for all preparations and assays. Potentiometric ments
and spectrophotometric
measure-
Potentiometric titrations were performed on the valine-, leucin* and isoleucinmpper complexes with metal : ligand ratio of 1: 1, 10e2 M concentration, 25”CandO.l M KN03.‘4Thedissociationconstants were calculated from the inflection points. With the copper complexes of valine, leucine and isoleucine ethylene-bridged compounds, dissolution does not occur until a slightly basic pH is reached. Rapid back-titration with HCI provided a means for titration to an acid pH before bulk precipitation occurred. The values obtained with the backtitration were used in the calculations. Titration of copper with the basic ligands for determination of stability constants were done as outlined for nickel glycine. l4 The calculation of association constants was adapted to a dibasic acid system as described by Courtney et al.’ ’ Equations (l)-(5) were assumed for the calculation of the dissociation constants where [L2 -1 is the concentration of completely deprotonated ligand, [LH-] is the concentration of monopronated ligand, [LH,] is the concentration of protonated
2033
Catalytic activity of ethylene-bridged aminoacid-copper(II) complexes NADH
ligand, and at- is the fraction of the deprotonated ligand : LH2 e LH- +H+,
(1)
LH- P L2- +H+,
(2)
k, = CLH-lCH+l D-b1 ’
(3)
klkz
z- -
aL
-
(5)
[H+]2+ki[H+]+k1k2’
The calculation of the metal-ligand speciation was done using eqns (6)-(S), where CL’-] = L, x a;-, CL=]is the original concentration and K1 and K2 are the association constants of metal and ligand : 1
B‘“‘+ = 1+K2[L2-]+KlK2[L2-]2
‘“t---2
&CL’-1 1+K,[L2-]+K,K2[L2-]2’
K,K2[L2-]2 =
1 +Kl[L2-]
M
’
fraction of CuHL+ ; (7) BCUL
IO IO-’
Fig. 1. The quenching of superoxide by the copper complexes using the NADH assay. (- ) Ethylenedileucine, (---) ethylene-di-isoleucine, and (--a-) ethylene-divaline.
fraction of free Cu2 + ; (6)
BcuLH+ =
4 6 6 Concentration
+K,K,[L’-1” fraction of CuL.
(8)
Calculations for speciation were made for a ligand concentration of 1 x 10m5 M similar to that for the superoxide-quenching assays. Calculations made for the alanine- and glycine-bridged copper complexes from the constants given by Chaberek and Martell16 indicate that both compounds contained less than 0.1% of the free Cu(I1). Calculations of metal-ligand association constants followed the procedure of Courtney et al. I5 for dibasic acids. All absorbance measurements were made with a Beckman 25 recording spectrophotometer at a pH of 7.6.
RIBULTS
The rates of reaction of the complexes with superoxide ion in vitro were leucine r alanine r 2methylalanine > isoleucine > valine >>glycine (cf. Figs l-4). The ligands without copper had no reactivity with the superoxide anion. Copper(I1) ion in the absence of the synthesized aminoacid ligands quenched superoxide at rates comparable to that of the leucine complex. The activity to quench the superoxide ion was examined at four different
K02
90
00 70 60 50
ESR spectroscopy
Copper complexes in aqueous solution buffered at pH 7 and ambient temperature were taken in capillaries with the concentration of the metal and ligand each at 10m3 M. Superoxide was prepared immediately before use as described above. Ratios of l:l, 1:2, 1:4, 1:6 and 1:8 (metal complex : superoxide) were prepared. Measurements were taken on a Model ER-420 JOEL X band ESR spectrometer.
40 30 20
2
4 6 6 Concentration
IO 10m6 M
Fig. 2. The quenching of superoxide by the copper Ethylene complexes using the KO, assay. (-) dileucine, (---) ethylene-d&isoleucine, (-.-) ethylenedivaline, and (. . .) Cuz+.
2034
B. M. KATZ and V. I. STENBERG NADH
I
I
I
2
I
I
I
4
6
8
Concentrotlon
2
3
4
5
IO 10-e
6
7
8
9
1011
PH
I
M
Fig. 3. The quenching of superoxide by the copper ) Ethylenecomplexes using the NADH assay. (ethylene-diglycine, and (-.-) dialanine, (---) ethylene-2-methylalanine.
Fig. 5. Species distribution for the 1: 1 copper(II)ethylerwdileucine complex as a function of pH. (a) Cuz+, (b) CuHL+, and (c) CuL.
IO_
0
9-
of the copper complexes. Although the reactivity order of the copper complexes were the same for the two independent assays, the potassium superoxide assay sensitivity decreased markedly at higher concentrations of the complexes. The results in Figs 5-7 indicate the speciation is the same for the three different copper complexes. The main species in the region of pH 7-8 for the valine-, leucine- and isoleucine-ethylene-copper complexes ia a copper complex with a net plus one charge. The titration curves of the ethylene-bridged amino acids (unprocessed data for Figs 5-7) shows .. two inflection points. concentrations
G 8E 2 u (;
765-
& 4ae 32II2
3
4
5
6
7
8
9
1011
PH
Fig. 6. Species distribution for the 1: 1 copper(IIb ethylendi-isoleucine complex as a function of pH. (a) Cu’+, (b) CuHL+, and (c) CUL.
2
4
6
Concentration
8
IO 10e6
M
Fig. 4. The quenching of superoxide by the copper ) Ethylenecomplexes using the KOs assay. (dialanine, and (--) ethylene-diglycine.
Fig. 7. Species distribution for the 1: 1 copper(II)ethylentiivaline complex as a function of pH. (a) Cu’+, (b) CuHL+, and (c) CuL.
Catalytic activity of ethylene-bridged aminoacid-copper(II) complexes
2035
Table 1. The UV and visible spectra of substituted ethylenediaminediacetic acid-copper complexes in aqueous solution Visible Copper-ethylene complex
1lppX (nm)
Valine Isoleucine Leucine Alanine Glycine 2-Methylalanine
635665 630-665 640-665 66&700 60&615 580620
uv
E,, 39 31 27 30 16 19
$4 254 250 250 250 nd” -*
E,, 4465 4135 3750 4000
‘Not detected. *Not determined. M
The visible and UV spectra from aqueous IOOG solutions of the bridged aminoacid-copper comFig. 8. ESR spectra of ambient aqueous solutions of plexes were determined in order to observe possible copper(I1) complexes. (a) Ethylene-N,N’-dialanine, (b) differences in configuration. The A,,,, and E,, are ethylenediglycine; (c) ethylen&V,N’-di-isoleucine, and summarized in Table 1. The visible region of the (d) ethylene-dileucine. spectra consists of broad absorption bands for all complexes. They are very close in energy but differ in intensity. The latter increase in the series : leucineDISCUSSION < isoleucine- < valine-copper complexes. The rZ, The substituted ethylenediaminediacetic acidfor the copper-alanine complex was slightly higher than the other copper complexes in the series. In the copper complexes formed from leucine, alanine and UV region of the spectra, very intense peaks at low 2-methylalanine display the highest rate of reactivity concentrations were observed at the 250~nm region with the superoxide ion of the derivatives for all complexes but the glycine-copper complex. synthesized (Figs l-4). The copper-glycine derivatThis peak was absent with copper and the ligands ive did not show any activity. In these complexes, the presence of the copper ion is essential to the without copper. complexes in order for them to react with the The ESR spectra of the copper-aminoacid bridged complexes were obtained in the presence superoxide anion. A superoxide intermediate is and absence of superoxide. All complexes except the believed to account for observations in kinetic glycine had four hyperfine peaks. The glycine one studies of superoxide reacting with copper comhad seven peaks. Figure 8 shows the spectra of plexes. 4~17 The site proposed for superoxide alanine-, glycine-, isoleucine- and leucint+copper attachment is the axial site of Cu(II), and the complexes before superoxide was added. The mechanism of the interaction is represented by eqns spectrum of the copper-alanine complex was poorly (9)-(11): resolved compared to the other copper complexes. Cu(I1) + 0; = Cu(II)O,, (9) After the first addition of superoxide anion, another type of spectra occurs which represents a cu(II)o; = Cu(I)O,, (10) paramagnetic species but differs from the spectrum Cu(I)O z*Cu(I)+O,. (11) without superoxide by an increase in hyperfine structure. This occurs before further reduction to a Cu(II)02 and Cu(I)02 are the oxygen adducts. species where all absorption bands disappear with The ESR spectra of solutions in which these further addition of superoxide. complexes are reacted with various concentrations The spectra for the copper-valine complex before of the superoxide anion give evidence for the initial and after addition of two increments of superoxide is bonding of the anion with the copper(I1) cation and illustrated in Fig. 9. The copper-valine complex its subsequent reduction to copper(I). The buffered, prior to superoxide addition did not have a aqueous, ambient-temperature ESR spectra of all pronounced hyperfine structure compared to the copper complexes show an initial increase in the leucine one (Figs 8 and 9). hyperflne structure of copper with increasing
2036
B. M. KATZ and V. I. STENBERG
c
I IOOG
Fig. 9. ESR spectra of ambient aqueous solutions of ethyleneN,N’-divaline with increments of superoxide. (a) No equivalent superoxide, (b) one equivalent superoxide, and (c) three equivalents of superoxide.
increments of superoxide followed by loss of the hyperflne structure as reduction to Cu(1) occurs. As an example, the ESR spectra of the valine derivative copper complex interaction with three different concentrations of superoxide are given in Fig. 9. In a study of copper salicylate, DeAlvare et d5 previously interpreted such spectra as superoxide complexing to copper with subsequent reduction. All five complexes examined herein have two nitrogen atoms complexed with the copper ion. Agarwal and Perrin I* have shown that the number of nitrogen atoms bound to copper can be distinguished by the visible spectra. If an absorption occurs below 600 nm, the copper ion is bound to three nitrogen atoms, but above 600 nm to two nitrogen atoms. The compounds in the present study all have absorption maxima above 600 nm (Table 1). The intense peaks in the UV spectra of the copper complexes are charge-transfer bands of the N-Cu-N bonds as verified by their absence in the spectra of copper solutions and the ligands without copper. By comparison with the bis(diamine)copper complexes, the bands are assigned to a c-o* (R,N-Cu-NR,) transition.” Only the glycine complex does not show the chargetransfer band. This indicates it has a different structure from the other four aminoacid complexes. Since the concentration of copper is identical for all the complexes, the data of Figs l-4 demonstrate that the copper ligand nature influences the rate of reaction with the superoxide anion. The coordination number of the copper ion does not change within the series: isoleucine, leucine, and valine ethylene-bridged copper complexes. The speciation
curves illustrated in Figs 5-7, calculated from the experimentally derived association constants for these three complexes, are virtually identical. Consequently, the diverse reactivities of these three complexes towards the superoxide ion are caused by factors other than speciation. Steric hindrance to the approach of the superoxide ion to the copper(I1) is believed to account for the decreased reactivity of the valine- and isoleucinebridged derivatives. Both have branching of the hydrocarbon chain at C-3, whereas the most reactive three complexes do not. Hence there is steric hindrance in close proximity to the active site. The 2methylalanine-bridged complex ability to quench superoxide approximates that of the leucinecomplex in spite of the extra methyl group at C-2. Therefore, the important stericinteractions appear to be at C-3. By using conformational analysis, Raos and Simeon2’ presented evidence that a methyl group which extends over copper reduces hydration of the axial copper site. This, together with the known tendency of copper complexes to distort toward tetrahedral, provides the possibility of the two C-3 alkyl groups of isoleucine and valine to extend over the axial site ofcopper to block this site and lessen its catalytic activity. 21-26 This was examined with the aid of molecular models. The C-3 alkyl groups of the isoleucine and valine complexes are unlikely to extend over copper in a square planar conformation. However, with an incease in distortion toward tetrahedral, these alkyl groups can readily be positioned over the copper(H). There is evidence for tetrahedral distortion among the six ethylenediamineacetic acid complexes of
Catalytic activity of ethylene-bridged aminoacid-copper(II) complexes copper studied herein. Red shifts and intensity enhancements of d-d electronic absorption bands accompany changes from square planar toward tetrahedral geometry.‘r The I, of the aminoacid bridged complexes (Table 1) have broad bands and are close in energy, but the intensities increase markedly from leucine- to valine-bridged copper complexes. With the exception of alanine, the degree of tetrahedral distortion roughly corresponds to the extent of superoxide quenching by these complexes. It appears that both the presence of two alkyl blocking groups at C-3 and tetrahedral distortion are necessary and act synergistically to block the axial site of copper(I1) to superoxide. Bereman et ~1.~’ concluded that tetrahedral distortion is the single most important dominating factor which changes Er12for their copper complexes and ESR parameters which reflect the degree of tetrahedral distortion varied linearly with E1,2.27 Yokoi and Addison” also showed that ESR parameters varied linearly with E,,,. It is therefore probable that the reduction potential varies in the series studied here. However, it appears that the resultant potential is a function of the structure which determines the availability of the axial copper site for the redox reaction with superoxide. The lack of activity of the copper glycine-bridged complex is attributed to formation of a dimer on the axial copper site. The complex is the smallest of the series, and small copper compounds have been found to dimerize.2g In an analogous study of copperaminoacid complexes, only with glycine did a third group add to the axial site of copper.30 Although dimeric copper compounds are antiferromagnetitally coupled, resulting in an absence of an ESR spectra,31 the ESR of the copper-glycine-ethylene
H H
1
‘c-c
H 1,H
2037
complex had seven peaks, indicating a weaker interaction of two copper nuclei (cf. Fig. 8). Although it was recognized that dimerization had occurred from a frozen ESR spectra of a 1: 1 copper-bridged glycine complex, the nature of the structure was not forwarded.32 The significance of seven lines for a copper dimer was attributed to out-ofplane interactions for the N,N’-ethylenebis(salicyldeneiminato) copper complex.33 Based on the ESR spectrum and the absence of a chargetransfer band which indicates a change of symmetry (Table l), a tentative structure is proposed (Fig. 10). The axial copper site in this structure is occupied by another bridged glycine so that it is inaccessible to super-oxide, resulting in deactivation of the catalytic activity of copper to quench superoxide.
REFERENCES 1. I. Fridovich, Science 1978,201,875. 2. G. Rotilio, A. Rigo and L. Calabrese, Frontiers in Physicochemical Biology, p. 357. AcademicPress, New York (1978). 3. J. Weinstein and B. M. Bielski,J. Am. Chem. Sot. 1980, 120,4916.
4. S. Goldstein and G. Czapski, J. Am. Chem. Sot. 1983, 105,7276.
5. L. R. DeAIvare, K. Goda and T. Kimura, Biochem. Biophys. Res. Commun. 1976,69,687.
6. C. Amar, E. Vilkas and J. Foos, J. Znorg. Biochem. 1982,17,313.
7. K. E. Joester, G. Jung, U. Weber and U. Weser, FEBS L&t. 1972,25,25.
8. F. Bell and K. A. K. Lott, Modern Approaches to Inorganic Chemistry, p. 150. Butterworths, London (1963). 9. R. J. Magee, W. Mazurek, M. J. CSConner and A. T. Phillip, Aust. J. Chem. 1974,27, 1885. 10. P. Blondeau, C. Berse and D. Gravel, Can. J. Chem. 1967,45,49. 11. N. Schlesinger, Ber. Chem. Dtsch. Ges. 1911,44,1135. 12. M. Younes and U. Weser, FEBS Lett. 1976,61,209. 13. R. Fried, Biochemie 1975,57,657. 14. R. J. Angelici, Synthesis and Technique in Inorganic Chemistry, p. 115. W. B. Saunders, Philadelphia (1977). 15. R. C. Courtney, S. Chaberek, Jr and A. E. Martell, J. Am. Chem. Sot. 1953,75,4814. 16. S. Chaberek, Jr and A. E. Martell, J. Am. Chem. Sot. 1952,74,5052. 17. J. Rabini, D. Klug-Roth and J. Lillie, J. Phys. Chem. 1973,77,1169.
l-i
-H
Fig. 10. Proposed structure for Cu(II)-e.thylenf+diglycine.
18. R. P. Agarwal and D. D. Perrin, J. Chem. Sot., Dalton Trans. 1976,89. 19. B. P. Kennedy and A. B. P. Lever, J. Am. Chem. Sot. 1973,%, 6907. 20. N. Raos and V. I. Simeon, J. Znorg. Biochem. 1983,1l#, 133.
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B. M. KATZ and V. I. STENBERG
21. K. F. Purcell and J. C. Kotz, Inorganic Chemistry, p. 761. W. B. Saunders, Philadelphia (1977). 22. R. C. Rosenberg, C. A. Root, R. K. Bernstein and H. B. Gray, J. Am. Chem. Sot. 1975,97,2092. 23. L. Sacconiand M. Ciampolini, J. Chem. Sot. 1964,276. 24. M. J. Adam and L. D. Hall, Can. J. Chem. 1982,60, 2229. 25. H. Yokoi, Bull. Chem. Sot. Jpn 1974,47,3037. 26. S. Yamada and A. Tokeuchi, Coord. Chem. Rev. 1982,43, 187. 27. R. D. Bereman, J. R. Dorfman, J. Bordner, P. D. Rillema, P. McCarthy and G. D. Shields, J. Znorg. Biochem. 1982,16,47.
28. H. Yokoi and A. W. Addison, Znorg. Gem. 1977,16, 1341. 29. R. bterberg, Coord. Chem. Rev. 1974,12,309. 30. I. Nagypti, E. Farkas and A. Gergely, J. Znorg. Nucl. Chem. 1975,37,2145. 31. U. Weser, K. H. Sellinger, E. Lengfelder, W. Werner and J. Striihle, Biochim. Biophys. Acta 1980, 631, 232. 32. N. Yokoi and M. Chikera, J. Chem. Sot., Chem. Commun. 1982,1125.
33. G. 0. Carlisle and W. E. Hatfield, Znorg. Nucl. Chem. Z.&t. 1970,6, 633.